A predicted ABC transporter, FtsEX, is needed for cell division in Escherichia coli - PubMed (original) (raw)
A predicted ABC transporter, FtsEX, is needed for cell division in Escherichia coli
Kari L Schmidt et al. J Bacteriol. 2004 Feb.
Abstract
FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria. Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial. Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX. RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop. We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl. We also found that in wild-type E. coli both FtsE and FtsX localize to the division site. Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI. Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not. Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent. We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media.
Figures
FIG. 1.
(A) Effect of salt and temperature on growth of RG60. RG60 (ftsE400::kan) growing in LB with 1% NaCl at 30°C was subcultured into LB with 1% (circles) or no (squares) NaCl at 30°C (open symbols) or 37°C (closed symbols). The inset shows a phase-contrast micrograph of cells harvested at the time indicated by the arrow from the cultures growing with salt. (B) Localization of FtsN. Cells of wild type (MG1655) or RG60 in exponential growth in LB with 1% NaCl at 30°C were fixed, and FtsN was visualized by immunofluorescence microscopy. Arrows point to septal localization of FtsN.
FIG. 2.
Effect of FtsEX depletion on growth and division. EC1335 (ftsE::kan/pBAD33-ftsEX) was grown in LB with no NaCl but containing either arabinose (closed symbols) or glucose (open symbols) to modulate expression of the plasmid-borne ftsEX genes. Samples were removed periodically to monitor growth by OD600 or cell morphology. The inset shows a phase-contrast micrograph of cells harvested at the time indicated by the arrow.
FIG. 3.
Localization of FtsE and FtsX to the division site. (A to D) Cells in exponential growth in LB with NaCl were fixed and examined by fluorescence microscopy directly (A and B), by indirect immunofluorescence microscopy (C and D), or by phase-contrast microscopy (C′ and D′). Strains shown are EC1063 (P204-ftsX-gfp) (A); EC1065 (P206-_gfp_-ftsX) (B); DHB4/pDSW609 (Plac-_ftsE_-3xHA) (C and D). (E) Relationship between cell length (age) and septal localization of FtsX. 509 cells of EC1063 were measured and scored for the presence (closed symbols) or absence (open symbols) of a fluorescent band at the midcell.
FIG. 4.
Effect of fts mutations on localization of FtsX to potential division sites in filamentous E. coli cells. Strains induced to express GFP-FtsX or FtsX-GFP were grown under nonpermissive conditions until they became filamentous, at which time they were fixed and examined by fluorescence microscopy to visualize GFP. Relevant division mutations are as follows: ftsZ84(Ts) in EC1158 (A), ftsA12(Ts) in EC1152 (B), and zipA1(Ts) in EC1340 (C); and FtsK depletion in EC1295 (D), FtsQ depletion in EC1179 (E), FtsW depletion in EC1159 (F), and FtsI depletion in EC1181 (G). The arrowhead in panel B points to a faint band sometimes observed in ftsA(Ts) filaments. 4′,6′-Diamidino-2-phenylindole staining was done to verify proper nucleoid segregation (data not shown).
FIG. 5.
Localization of various division proteins after depletion of FtsEX. The strains used express ftsEX under control of an arabinose-dependent promoter and harbor gfp fusions to different division genes. These strains were grown in parallel in media containing arabinose (short cells) or glucose (filaments) and then fixed and examined by fluorescence microscopy to visualize GFP. (A and B) FtsA-GFP in EC1363; (C and D) ZipA-GFP in EC1391; (E and F) FtsK (1-266)-GFP in EC1386; (G and H) GFP-FtsQ in EC1392; (I and J) GFP-FtsI in EC1366. 4′,6′-Diamidino-2-phenylindole staining was done to verify nucleoid segregation (data not shown).
FIG. 6.
Model for recruitment of proteins to the septal ring. The first event is polymerization of FtsZ into the Z-ring. FtsA, ZipA, and ZapA bind directly to FtsZ and localize next or concomitantly with Z-ring assembly. Once either FtsA or ZipA has joined the septal ring, the remaining proteins localize in the order indicated. Whether any E. coli proteins are dependent upon ZapA is not yet known.
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